Efficacy of linezolid in the treatment of mediastinitis
due to methicillin-resistant Staphylococcus aureus:
an experimental study
Mustafa Sacar
a,*
, Suzan Sacar
b, Ilknur Kaleli
c, Nural Cevahir
c,
Zafer Teke
d, Semra Toprak Kavas
b, Ali Asan
b, Faruk Onder Aytekin
d,
Ahmet Baltalarli
a, Huseyin Turgut
baDepartment of Cardiovascular Surgery, Pamukkale University Faculty of Medicine, Kinikli Kampusu, Kinikli 20070 Denizli, Turkey b
Department of Clinical Microbiology and Infectious Diseases, Pamukkale University Faculty of Medicine, Denizli, Turkey
c
Department of Microbiology, Pamukkale University Faculty of Medicine, Denizli, Turkey
dDepartment of General Surgery, Pamukkale University Faculty of Medicine, Denizli, Turkey
Received 18 June 2007; received in revised form 25 August 2007; accepted 17 September 2007 Corresponding Editor: Craig Lee, Ottawa, Canada
http://intl.elsevierhealth.com/journals/ijid
KEYWORDS
Linezolid;
Surgical site infection
Summary
Introduction: The treatment of postoperative mediastinitis is very important because of its high morbidity, mortality, and increased hospital stay and hospital costs. The aims of our research were to investigate whether linezolid alone can be an effective treatment agent for methicillin-resistant Staphylococcus aureus (MRSA) mediastinitis, and to determine whether linezolid can provide synergistic activity when given in combination with rifampin.
Methods: A partial upper median sternotomy was performed on 70 rats. The animals were divided into seven groups: an uncontaminated control group; an untreated contaminated group; three contaminated groups that received antibiotic therapy with either 25 or 50 mg/kg linezolid twice a day, or rifampin 5 mg/kg twice a day; and two contaminated groups that received a combination therapy consisting of 25 or 50 mg/kg linezolid and rifampin 5 mg/kg twice a day. The antibiotic treatment lasted 7 days. Tissue samples from the upper ends of the sternum and swab specimens of the upper mediastinum were obtained and evaluated microbiologically.
Results: The 25-mg/kg dose of linezolid, either alone or combined with rifampin, was not effective in reducing the bacterial counts in mediastinum and sternum. Quantitative bacterial cultures of mediastinum and sternum were significantly lower in the groups receiving 50 mg/kg linezolid alone or in combination with rifampin compared with the control. Adding of rifampin to linezolid therapy did not result in a significant change in bacterial counts versus linezolid alone. Conclusion: A high dose of linezolid should be considered as a possible therapeutic agent for the treatment of post-sternotomy infection caused by MRSA.
#2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +90 258 2118585x2251; fax: +90 258 2410040. E-mail address:[email protected](M. Sacar).
1201-9712/$32.00 # 2007 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijid.2007.09.017
Introduction
Although postoperative mediastinitis after open heart sur-gery is a rare complication, it is associated with a mortality of up to 47% and increased hospital stay and hospital costs.1—3 Staphylococci are the main causative organisms, accounting for 54—79% of cases.1,4Staphylococcus aureus mediastinitis often occurs due to perioperative contamination of the mediastinal space.2Methicillin-resistant Staphylococcus aur-eus (MRSA) mediastinitis has been found to be associated with higher rates of overall mortality, mediastinitis-related death, and treatment failure.5
Optimal outcomes in post-surgical mediastinitis require surgical management in addition to systemic antibiotic ther-apy.6—8MRSA is showing an increasing prevalence worldwide
and most strains are now also resistant to fluoroquinolones, macrolides, tetracyclines, and aminoglycosides.9,10Since the antibiotics used traditionally for the treatment of serious infections caused by these resistant staphylococci are lim-ited, there is a requirement for new therapeutic approaches. Linezolid is a synthetic oxazolidinone antimicrobial agent that has a good activity against most medically important Gram-positive microorganisms resistant to methicillin and vancomycin.11,12Recently dual or multi-drug combinations
have been encouraged in the hope of preventing antibiotic resistance.13,14Rifampin is highly bactericidal towards Sta-phylococcus species.15Several trials of linezolid, alone or in combination with rifampin, have been published;16—19 how-ever, its efficacy and safety in mediastinitis are less well reported.
The aims of our research were to investigate whether linezolid alone can be an effective treatment agent for MRSA mediastinitis, and to determine whether linezolid can pro-vide synergistic activity when given in combination with rifampin.
Methods
This study was approved by the local animal research ethics committee. During the entire study the animals were kept at our institution’s animal research laboratory under veterinary supervision. All animals received human care in compliance with Principles of laboratory animal care, formulated by the Guide for the care and use of laboratory animals, prepared by the National Academy of Sciences.20
Organisms and susceptibility testing
The strain of MRSA used in this study was isolated from blood culture and a surgical site infection specimen from a patient with post-sternotomy mediastinitis following open heart sur-gery. Identification of the clinical isolates was determined by Gram staining, catalase reaction, tube coagulation test, and Api-Staph test (bioMe´rieux, Lyon, France). Methicillin sensi-tivity was investigated by oxacillin disk diffusion test.21
The antimicrobial susceptibility of the strain was deter-mined by using the broth microdilution method, according to the procedures outlined by the Clinical and Laboratory Stan-dards Institute specifications.22,23The minimum inhibitory concentration was taken to be the lowest antibiotic concen-tration at which observable growth was inhibited.
Drugs
Linezolid (Pfizer, Halden, Norway) and rifampin (Kocak Farma, Istanbul, Turkey) were diluted in accordance with the manufacturers’ recommendations. Solutions of drugs were made fresh on the day of assay.
In vivo rat model
Seventy adult male Wistar rats weighing between 300 and 350 g were randomly divided into seven groups. There were 10 rats in each group. Group 1 was an uncontaminated control group with no contamination and no antibiotic therapy given; group 2 was an untreated contaminated control group with local MRSA contamination and no antibiotic therapy given; group 3 consisted of those with local MRSA contamination given intraperitoneal linezolid (25 mg/kg of body weight) twice a day; group 4 consisted of those with local MRSA contamination given intraperitoneal linezolid (50 mg/kg of body weight) twice a day; group 5 consisted of those with local MRSA contamination given intramuscular rifampin (5 mg/kg of body weight) twice a day; group 6 consisted of those with local MRSA contamination given intraperitoneal linezolid (25 mg/kg of body weight) and intramuscular rifam-pin (5 mg/kg of body weight) twice a day; group 7 consisted of those with local MRSA contamination given intraperitoneal linezolid (50 mg/kg of body weight) and intramuscular rifam-pin (5 mg/kg of body weight) twice a day.
Each rat was anesthetized intramuscularly with a 2:1 mixture of ketamine hydrochloride (100 mg/ml; Pfizer) and xylazine hydrochloride (20 mg/ml; Bayer) at a dose of 0.75 ml/kg. The hair on the rats’ chests was shaved, the skin cleaned with 10% povidone-iodine solution, and a sterile towel was used to cover the sternum. The skin and presternal layers were incised. A partial upper median sternotomy without access to pleural spaces was performed on all rats. Groups 2—7 were inoculated with 0.5 ml 108colony-forming units (CFU)/ml MRSA in the mediastinal and in the sternal layers. The sternum and the presternal layers were closed with a single 3-0 Prolene suture. Animals were returned to individual cages and examined thoroughly daily. During the treatment period no medication was given to the rats in groups 1 and 2. Postoperatively, groups 3—7 were given antibiotic treatment for seven days, twice daily, starting 24 hours after the end of the procedure. After 12 hours following the end of treatment, the rats were sacrificed, a sternotomy was performed for each rat, and tissue samples from the upper ends of the sternum and swab specimens of the upper mediastinum were aseptically obtained.
Assessment of the infection
Mediastinal swab specimens were placed in tubes containing 1 ml of phosphate-buffered saline solution and sonicated for 5 minutes. Quantitation of viable bacteria was performed by culturing serial 10-fold dilutions (0.1 ml) of the bacterial suspension on blood agar plates. All plates were incubated at 37 8C for 48 hours and evaluated for the presence of the staphylococcal strains. The organisms were quantitated by counting the number of CFU per plate. Tissue samples were weighed and homogenized under sterile conditions in 1 ml of sterile phosphate-buffered saline. The serial diluted
genates were inoculated (0.01) onto blood agar and incu-bated at 37 8C. After 48 hours, the amount of growing bac-teria was determined as CFU/g tissue.
Statistical analysis
Quantitative culture results were presented as arithmetic mean standard deviation (SD) of CFU. Differences among the groups were evaluated using one-way analysis of variance (ANOVA), and multiple comparisons between the groups were performed with a posthoc test (Tukey’s HSD test). Differences were considered statistically significant when p < 0.05. Data were analyzed using statistical software SPSS for Windows 11.0 (SPSS, Chicago, IL, USA).
Results
According to the broth microdilution method, the clinical isolate was susceptible to linezolid and rifampin. The mini-mal inhibitory concentrations (MICs) for linezolid and rifam-pin in the strain of S. aureus used in this study were found to be 2 mg/ml and 0.5 mg/ml, respectively.
Evidence of mediastinitis was present in all rats of the untreated contaminated group. In contrast, none of the animals included in the uncontaminated control group had anatomic or microbiological evidence of mediastinitis. All the rats in the groups that received antibiotic treatment, except two rats in group 4 and a rat in group 7, showed evidence of infection. Culture negative rates and bacterial counts for each group are shown inTable 1.
The average growth of the microorganisms in the med-iastinum and sternum were compared. The 25-mg/kg dose of linezolid, either alone or combined with rifampin, was not effective in reducing the bacterial counts in mediastinum and sternum in comparison with the contaminated untreated group ( p > 0.05). Quantitative bacterial cultures of medias-tinum and sternum were significantly lower in the groups receiving 50 mg/kg linezolid alone or in combination with rifampin (groups 4 and 7) when compared to the untreated
contaminated group (group 2) and groups receiving 25 mg/kg linezolid, rifampin, or their combination (groups 3, 5, and 6; p < 0.05). However, there was no statistically significant difference in terms of mean bacterial counts between the 25 mg/kg linezolid monotherapy group and the 25 mg/kg linezolid plus rifampin group (group 6; p > 0.05). The same difference was also found between the 50 mg/kg linezolid monotherapy and rifampin combination groups. Finally, there was no mortality among the rats and we did not encounter any clinical evidence of drug-related adverse effects, such as local signs of perigraft inflammation, anor-exia, vomiting, diarrhea, or alteration in behavior.
Discussion
In this study of experimental mediastinitis caused by MRSA, treatment with linezolid at 50 mg/kg was found to be effec-tive in reducing the bacterial count in mediastinum and sternum, whereas the same efficacy was not observed at 25 mg/kg. The combination of linezolid and rifampin was not able to significantly change the treatment results when compared with those obtained with linezolid alone.
There are reports of several clinical and experimental studies of linezolid alone or in combination with rifam-pin;16,17,24 however, its exact efficacy and safety in post-sternotomy mediastinitis have not been well reported. Jac-queline et al.25 found that linezolid plus rifampin was the most active combination against MRSA strains in comparison with gentamicin and vancomycin in time—kill experiments. In vivo studies of the linezolid—rifampin combination in an MRSA bacteremia model and a methicillin-sensitive S. aureus (MSSA) endocarditis model have shown additivity or indiffer-ence.18,19 The purpose of our study was to evaluate the efficacy of linezolid as a treatment agent for MRSA medias-tinitis and to investigate whether linezolid can provide synergistic activity when given in combination with rifampin in the treatment of that serious infection. Based on prior experimental and clinical research on MRSA infections,18,26—
29we developed an experimental mediastinitis model.
Table 1 Outcome of 7-day treatment of experimental mediastinitis caused by methicillin-resistant Staphylococcus aureus (MRSA)
Group Drug and dose No. culture
negative/total
Mean bacterial count
Swab Sternum Mediastinuma Sternumb
Group 1 (Uncontaminated control) No drug 10/10 10/10 0.0 0.0 Group 2 (Untreated contaminated control) No drug 0/10 0/10 5.20 0.15 5.59 0.20 Group 3 Linezolidc(25) 0/10 0/10 4.68 0.28 5.18 0.20 Group 4 Linezolidc(50) 2/10 2/10 2.49 1.32e 2.59 1.38f Group 5 Rifampind(5) 0/10 0/10 4.87 0.07 5.25 0.20
Group 6 Linezolidc(25) + rifampind(5) 0/10 0/10 4.85 0.10 5.21 0.19
Group 7 Linezolidc(50) + rifampind(5) 1/10 1/10 2.97 1.05e 3.06 1.08f
aLog
10CFU/ml SD. bLog
10CFU/g SD.
c Administered intraperitoneally twice daily. dAdministered intramuscularly twice daily.
ep < 0.05 vs. the mean bacterial counts of the mediastinum in groups 2, 3, 5, and 6. f p < 0.05 vs. the mean bacterial counts of the sternum in groups 2, 3, 5, and 6.
In recent years the effectiveness of new antimicrobial agents has been widely investigated in cardiovascular infec-tions. The increasing prevalence of MRSA in cardiac surgery units has led to increased vancomycin utilization for these serious and life-threatening infections seen in these units.30—
34 Since the emergence of glycopeptide intermediate and
resistant S. aureus,35,36 clinicians have understood the importance of minimizing glycopeptide use.
In order to choose a proper treatment and prevent unne-cessary use of broad-spectrum antibiotics, an exact defini-tion of post-sternotomy mediastinitis should rely on both clinical and bacteriological criteria. This is especially impor-tant with regard to infections caused by MRSA, as these infections have a comparatively worse clinical outcome.5,37 Linezolid has been demonstrated to be effective in the clinical management of MRSA infections.23,38,39We investi-gated the activity of linezolid in an experimental model of mediastinitis due to MRSA, because analogous infections in humans are common in clinical practice and generally require aggressive surgical debridement and prolonged courses of systemic antimicrobial treatment.
The dose of linezolid was selected to mimic the thera-peutic dose given to patients, and was also based on dose regimens used in other animal models of infection. A 25-mg/ kg oral dose of linezolid in rats produces plasma exposures comparable to those obtained in humans following a 600-mg oral dose.40—42 The approximately 100% bioavailability of linezolid allows the drug to be administered either parent-erally or orally in the same dose without requiring a dose adjustment.43 The excellent bioavailability of linezolid in rats has also been demonstrated.28
We used a normal dose and a higher dose, as our MRSA strain was less susceptible in vitro. For the same reason Jabes et al.42used linezolid at 50-, 100-, and 200-mg/kg doses once orally in rats infected with MRSA. Based on the pharmacoki-netics of linezolid against the strain tested, they expected the dose of 50 mg/kg to be effective against MRSA. But this dose was found to be ineffective in reducing the bacterial load of the MRSA strain. A 100-mg/kg oral dose of linezolid was found to be necessary to achieve an adequate reduction in bacterial titer.
Oramas-Shirey et al.44reported that a 25-mg/kg intrave-nous dose of linezolid given three times a day for 5 days was not effective in the treatment of experimental staphylococ-cal endocarditis in a rabbit model, whereas doses of 50 or 75 mg/kg with the same treatment schedule were found to be effective.
The efficacy of linezolid at 50 mg/kg/day or 100 mg/kg/ day in a rat model of pneumococcal pneumonia has also been evaluated, and the latter was found to be more effective.25 As linezolid has not been extensively investigated in cardi-ovascular infections, and there have been reports of treat-ment failure in another serious cardiovascular infection endocarditis with the standard dose used in humans,45 we
decided to investigate the efficacy of linezolid at a double dose in mediastinitis and compare it with the standard dose. We found that linezolid at a double dose was effective in reducing the bacterial count in mediastinum and sternum. We did not observe any side effects with the high dose, but this requires confirmation in future studies in humans.
Additionally, our results demonstrated that adding rifam-pin to linezolid treatment did not change the results obtained
with linezolid alone. This is especially important in practice, as clinicians should also minimize rifampin utilization in order to avoid rifampin resistance.
Linezolid at 50 mg/kg seemed to be an effective agent in our experimental mediastinitis model, and this result sug-gests that this antibiotic may be a reasonable therapeutic choice. Unfortunately, although the possibility of acquired resistance to linezolid is low, there are recent studies report-ing a risk of linezolid resistance after exposure to this drug.46—48 This is why it should be considered only for patients with serious MRSA infections that are poorly respon-sive to the glycopeptides or those who do not tolerate glycopeptide treatment.
More clinical studies are required on the combination of linezolid with other antimicrobial agents in order to increase the bactericidal activity of therapy, to prevent the emer-gence of drug-resistant subpopulations, and to provide a complementary antibacterial spectrum.
When considering our results, the following limitations of our study need to be taken into account. Our in vivo model used a direct form of MRSA inoculation in the mediastinal and in the sternal layers. Additionally, we did not determine serum antibiotic concentration in the rats. Unfortunately, the required technical equipment for measuring the anti-biotic concentration is not present in our hospital as we live in a country with limited financial resources. We used linezolid in a dose that mimics the validated human dose and its double form, and investigated its efficacy by evaluating the presence and the amount of the staphylococcal strains in the infected area. However, the animal model in this study is not directly comparable with the real situation in a living human being. In conclusion, linezolid at 25 mg/kg, rifampin, and the combination of linezolid at 25 mg/kg and rifampin did not significantly reduce the bacterial counts and were found to be ineffective in the treatment of our experimental medias-tinitis model. The combination of rifampin with linezolid therapy did not result in a significant change in bacterial counts versus linezolid alone. Finally, increasing the dose of linezolid from 25 mg/kg to 50 mg/kg resulted in a significant reduction in bacterial counts. Based on our findings, high-dose linezolid should be considered as a possible therapeutic agent for the treatment of post-sternotomy infection caused by MRSA. Our results need to be confirmed in further pre-clinical and pre-clinical studies.
Conflict of interest: No conflict of interest to declare.
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